Mohammad et al. The Journal of Basic and Applied Zoology (2021) 82:34 The Journal of Basic https://doi.org/10.1186/s41936-021-00214-1 and Applied Zoology

RESEARCH Open Access The toxic effect of zinc oxide nanoparticles on the terrestrial nyctelia (-) Wafaa A. Mohammad1, Safaa M. Ali2* , Nasser Farhan3 and Shimaa Mohamed Said2

Abstract Background: Chemicals have deleterious effect on the environment. The wide use of nanomaterials as products for plant protection, fertilizers, and also in water purification leads to the release of these materials to the environment. Terrestrial gastropods including snails and have the ability to accumulate heavy metals in their bodies. The present study evaluates the toxic effect of zinc oxide nanoparticles on the terrestrial slug Lehmannia nyctelia. Zinc oxide nanoparticles (ZnO NPs) were prepared by thermal decomposition method. ZnO NPs are characterized by X- ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and ultraviolet-visible spectroscopy (UV). Slugs were treated with three different concentrations of ZnO NPs. Results: A total of three died by the end of the experiment. Many histological alterations were detected after exposure to different concentration of ZnO NPs. Conclusions: The obtained histological alterations prove the toxic effect of ZnO NPs on the under study.

Background effects such as toxicity of non-target organisms, contam- Molluscs are considered as one of the important animals ination of food and environment (Jeong et al., 2012). in the food chain and noticed as agricultural pests. Toxic Terrestrial gastropods are one of the most efficient ac- chemicals produce harmful effect on terrestrial gastro- cumulators of metals and they are considered as bio- pods at the cellular level (Boer et al., 1995; Hernadi indicators to environmental pollution (Pihan & de Vau- et al., 1992). At the same time, they possess special fleury, 2000; Snyman et al., 2005; Viard et al., 2004). Ter- mechanisms to get rid of several harmful chemicals restrial snails and slugs have a great capacity of the which accumulated in their tissues (Ferner, 2001). accumulation and magnification of heavy metals and are Slugs are considered as a great threat to many of the considered important to monitor the bioavail- temperate horticultural crops. Seedlings are very sensi- ability of metallic components in the soil if they com- tive to slug as if they cause a little amounts of damage to pared to other invertebrates (Coeurdassier et al., 2000; the growing tips of seedlings, this will lead to plant death Lanno & Mc Carty, 1997; Wensen et al., 1994). (Moens & Glen, 2002; Nash et al., 2007). Recently, the industrial development in agricultural Slug Lehmannia valentiana is a pest which result in field increased rapidly and leads to inorganic and organic many troubles to greenhouse. The common method to contamination from harmful heavy metals and chemicals control this slug is the chemical molluscicides. The to the terrestrial and aquatic ecosystems. These chemi- chemical molluscicides may cause undesirable side cals show deleterious effect on aquatic and terrestrial en- vironment (Davidson et al., 2011; Pack et al., 2014; Sanchez, 2008).

* Correspondence: [email protected] Nanomaterials were widely used in agriculture during 2Zoology Department, Faculty of Science, Assiut University, Assiut, Egypt the last 10 years (Buzea & Pacheco, 2017; Gogos et al., Full list of author information is available at the end of the article

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2012). They were using as plant protection products, fer- applying Scherrer’s equation: D =Kλ/βcosθ, where K is tilizers, water purification, for soil improvement, and the shape factor whose value is taken as 0.89, λ is the pollutant remediation (Parisi et al., 2015). In many cases, wavelength of Cu Kα radiation (λ = 1.54178), and β is nanoparticles (NPs) are applied to the soil directly as a the corrected full width at half maximum (FWHM) of fertilizer or in the form of an aerosol (Sturikova et al., the diffraction peak and θ is the diffracting angle. 2018) while seeds are mostly soaked in aqueous NP sus- pension (Lin & Xing, 2007; Segatto et al., 2018). In the Fourier transform infrared spectroscopy (FTIR): future, technological application of NPs may be un- Infrared spectra were recorded on a Thermo Fisher– desired as they are released to the environment and dif- model: Nicolet iS10 FTIR spectrometer in a wave num- − fused in the soil due to their small size (Kaegi et al., ber range 4000–500 cm 1. 2008). Zinc oxide nanoparticles (ZnO nanoparticles) are in- SEM and TEM teresting materials due to their unique electrical and op- The morphology of the ZnO NPs powder sample was in- tical properties, their wide-band semiconductor, and vestigated by scanning electron microscopy (SEM; JEOL they are suitable for many applications (Mishra et al., [model: JSM 5400LV]) and transmission electron mi- 2011). ZnO NPs are useful in environmental remediation croscopy (TEM; JEOL [model: JEM-100 CXII]). due to their high capabilities for degradation and elimin- ation of pollutants in air or water due to their strong UV-visible spectroscopy physical adsorption and their chemical catalytic proper- The UV-vis absorption measurements of ZnO NPs were ties (Jing et al., 2001). Zn ONPs are used as antimicro- recorded in the range 200–800 nm by PerkinElmer; bial agent, and also used in cosmetics, sunscreens, and model: LAMBDA 750 UV/Vis/NIR Spectrophotometer. coatings (Ali et al., 2012). Due to the increasing use in The synthesized ZnO NPs powder sample was dispersed consumer products, ZnO NPs will be found in the at- in ethanol and optical characterizations were executed. mospheric, terrestrial, and aquatic environments (Ali et al., 2012; Sales, 2013; Service, R.F., 2008). Collection of specimens The present study aimed to evaluate the toxic effect of The experimental work was carried out using specimens ZnO NPs on theterrestrial slug Lehmannia nyctelia. of Lehmannia nyctelia, collected during autumn from Assiut University farm, Assiut Governorate, Egypt. Slugs Methods were kept in normal laboratory conditions, maintained Chemicals and reagents in plastic containers containing soil obtained from their All chemical and reagents purchased were analytical natural habitat and fed with fresh lettuce daily. grade. Zinc nitrate hydrate was purchased from Alfa Aesar (Ward Hill, MA), Ethanol, abs. 100% a.r. was pro- Experimental design and ZnO NPs exposure cured from Chem-Lab., Belgium. For the experiment, the acclimatized healthy slugs with an equal body weight (0.6 gm) were randomly distrib- Preparation of ZnO nanoparticles uted into four groups (control and three treated groups). The zinc oxide nanoparticles (ZnO NPs) were prepared Each group consists of 10 individuals and was kept in by thermal decomposition method as described by Shan- plastic containers containing the same weight of soil and kar et al. (2013). Three grams of zinc nitrate were taken each container covered with perforated cloth for ventila- and fired in a furnace (air atmosphere) for 3 h at 500 °C. tion. The control slug’s group fed on fresh lettuce It was further grinded in a mortar to make a fine powder impressed in 10 ml distilled water. of ZnO NPs. However, the treated groups were fed on fresh lettuce impressed in different concentrations of ZnO NPs (0.01 Characterization of ZnO NPs g/10 ml dw, 0.02/10 ml dw, 0.05/10 ml dw, modified ZnO NPs were characterized by X-ray diffraction (XRD), from Fahmy and Abdel-Ghaffar (2014). Exposure period Fourier transform infrared spectroscopy (FTIR), scan- was 10 days, during which the slugs were subjected twice ning electron microscopy (SEM), and ultraviolet-visible to the contaminated food with the previously mentioned spectroscopy (UV). concentrations of ZnO NPs. At the end of the experi- mental period, surviving slugs were used for histological X-ray diffraction (XRD) studies. Died animals were counted and removed. The structure of the nanoparticles was investigated by a Slugs were fixed in neutral formalin and prepared for Philips X-ray diffractometer (model PW 1720) at room histological study. Seven micrometers transverse sections temperature, with a step of 0.06°, in the range of 4° ≤ 2θ were prepared and stained with hematoxilin and eosin ≤ 89.89°. The average crystallite sizes were calculated by (HE). Sections were examined by light microscope. The Mohammad et al. The Journal of Basic and Applied Zoology (2021) 82:34 Page 3 of 9

organs under study are the digestive gland, salivary while Figure 2b presents a typical TEM image of ZnO glands, and hermaphroditic glands. NPs. It is clear that in this case a ZnO NPs was success- fully prepared. Results Characterization of ZnO nanoparticle The UV-Vis spectrum X-ray diffraction (XRD) The UV-Vis spectrum of ZnO NPs is shown in Fig. 2c. The ZnO NPs were characterized by X-ray diffraction Confirmation of the synthesized ZnO product in nano- (XRD). A definite line broadening of the XRD peaks in- scale was exhibited by the highly red shifted absorption dicates that the prepared material consists of particles in maximum occurring around 400 nm (Datta et al., 2017). the nanoscale range (Fig. 1a). All the diffraction peaks No other peaks were observed in the spectrum, confirm- can be indexed to the ZnO hexagonal wurzite structure ing that the synthesized product was ZnO only. (JPCDS card number: 04-008-8196). The average particle size of the sample was found to be 21 nm which is de- Results of histology rived from the full width at half-maximum (FWHM) of At the end of the experiments, three animals died; one more intense peak corresponding to (Shankar et al., after exposure to concentration 0.02 g/10 ml dw ZnO 2013) plane located at 36.34° using Scherrer’s formula. NPs and two died after exposure to 0.05 g/10 ml dw ZnO NPs.

FTIR analysis Histological observation of the digestive gland (Fig. 3a–d) Figure 1b shows the FTIR spectrum of the zinc oxide −1 nanoparticles. The special peak at 500 cm owing to Untreated slugs The digestive gland of the slug com- Zn-O vibrational mode (18-19); another strong band at −1 poses of lobes. Each lobe composes of large number of 1013 cm may be assigned to the stretching and bend- digestive tubules separated with loose connective tissue. ing vibrational modes of the Zn-O bonds, respectively. Each tubule is surrounded externally with circular muscle layer. The digestive acini are lined with simple Electron microscopic investigation of ZnO NPs epithelial cells which arranged around narrow irregular The morphology of ZnO NPs was characterized by SEM lumen (Fig. 3a). and TEM techniques. Figure 2a shows the SEM image; There are four different cell types were observed lining the digestive gland tubules. The two main cell types are digestive and calcium cells. The third type of cells is ex- cretory cells, while the fourth type comprises thin cells which distribute randomly between the different cells. These cells have the following characteristic features: Digestive cells They are the most abundant cell type in the digestive gland tubule epithelium. These cells are observed as a simple columnar epithelium with flattened or slightly broad apical surfaces and narrow base. They appear under the light microscope with highly vacuolated cytoplasm. Calcium cells These cells are fewer than digestive cells. They are pyr- amidal in shape with narrow distal end and broad base. Calcium cells are basophilic cells, mostly have large rounded nuclei. Excretory cells They are basophilic cells and globular in shape. Thin cells These cells are narrow extending to the full height of the epithelium.

Fig. 1 a X-ray diffraction patterns of ZnO NPs. The peaks refer to the Slugs exposed to 0.01 g/10 ml dw ZnO NPs The epi- Wurtzite hexagonal structure in the reference data (JCPDS No. 04- thelial cells became shorter. Some of them are ruptured 008-8196). b FTIR Spectra of ZnO NPs and lost their content. The lumen in some acini of Mohammad et al. The Journal of Basic and Applied Zoology (2021) 82:34 Page 4 of 9

Fig. 2 a SEM image of ZnO NPs. b TEM image of ZnO NPs. c UV-Vis analysis of ZnO NPs digestive gland increased in size and filled with the cells Many cells lost their contents and the vacuolated cells remains (Fig. 3b). increased in number.

Slugs exposed to 0.02 g/10 ml dw ZnO NPs Some di- Slug exposed to 0.02 g/10 ml dw ZnO NPs Many gestive tubules were separated from the surrounding histological changes were observed (Fig. 4c). Most of the muscle layer. Many cells are ruptured and lost their con- acidophilic and vacuolated cells lost their shapes and tents. Large vacuoles are detected in many cells. The their nuclei became smaller in size, flattened, and mar- lumen of the acini became narrow (Fig. 3c). ginal in position. Large spaces were appeared among dif- ferent cell types. Slugs exposed to 0.05 g/10 ml dw ZnO NPs: The con- nective tissue among acini is destroyed leaving large Slug exposed to 0.05 g/10 ml dw ZnO NPs Acido- empty spaces. Most of the epithelial cells were rup- philic cells increased in number and most of them be- tured. The acini became narrow and contain cells re- came smaller in size, with flattened and marginal nuclei mains (Fig. 3d). (Fig. 4d). Many vacuolated cells lost their content and became empty cells. Histological observations on the salivary gland: Histological observation of gonads: (Fig. 5a–d) Untreated slugs (Fig. 4a) The examination of the un- treated slug’s salivary gland showed that it consists of Untreated slugs The hermaphroditic gland of the slug three types of glandular cells. These types showed great is composed of a small number of lobes; each lobe con- variation in their contents and staining properties; type sists of various acini separated by thin interstitial con- (I) has purple color, type (II) has a blue color and type nective tissue and enveloped by squamous epithelial (III) is a vacuolated cell. cells (Fig. 5a). Walls of the acini are lined with clusters of germinal epithelial cells and all acini contain different Slugs exposed to 0.01 g/10 ml dw ZnO NPs The saliv- stages of developed cell as a result of both oogenesis and ary gland showed many histological changes (Fig. 4b). spermatogenesis. Mohammad et al. The Journal of Basic and Applied Zoology (2021) 82:34 Page 5 of 9

Fig. 3 Photomicrograph of sections through the digestive gland of a untreated slugs showing: connective tissue (CT), digestive cells (DC), thin cells (TC), calcium cells (CC), and lumen (L). b Slugs treated with 0.01 ZnO NPs showing: large lumen (L), shorter cells (SC), and ruptured cells (RC). c Slugs treated with 0.02 ZnO NPs showing: vacuoles(V), ruptured cells (RC), and separated muscle (arrows). d Slugs treated with 0.05 ZnO NPs showing: remains cells (arrows), vacuolated cells (VC), ruptured cells (RC), and space (S)

Slug exposed to 0.01 g/10 ml dw ZnO NPs Degenera- (Merdzan et al., 2014; Romero-Freire et al., 2017). It also tive changes were observed in most of different stages of uses as antibacterial agent and fertilizer (Ma et al., 2013; gametogenesis (Fig. 5b). The squamous epithelial layer Parisi et al., 2015; Segatto et al., 2018). that surrounded the acini is ruptured in some places. Results obtained from infrared spectra that recorded Large empty spaces appeared among the acini. Blood in the present study showed that the special peak at 500 − cells infiltration is detected. Some sperms are cm 1 owing to Zn–O vibrational mode(18-19); another − degenerated. strong band at 1013 cm 1 may be assigned to the stretching and bending vibrational modes of the Zn–O Slug exposed to 0.02 g/10 ml dw ZnO NPs Signs of bonds, respectively (Kumar et al., 2015; Punnoose et al., degeneration in different gametogenesis stages were de- 2014). The FTIR spectra of ZnO NPs showed that the tected. They were represented by fibrosis and appear- high purity of ZnO NPs was prepared by thermal de- ance of many spaces in the acini lumen, in addition to composition method. appearing large spaces among acini (Fig. 5c). The present histological investigations of the digest- ive gland of land slug Lehmannia nyctelia confirm Slug exposed to 0.05 g/10 ml dw ZnO NPs The early the existence of four cell types: digestive, calcium, ex- gametogenesis stages increased in number and aggre- cretory, and thin cells. This finding is agreed with re- gated in clusters (Fig. 5d). Many of the gamete’s stages sults of Hamed et al., 2007;AboBakr(2011)and lost their shapes and degenerated. Fibrosis in the lumen Yousef et al. (2011) in the land snail, Eobania Vermi- of acini was increased. culat. Sharaf et al. (2015) as well as Mustafa and Awad (2018) revealed that the digestive gland is con- Discussion sisted of three types of cells in the land snail Heli- Land snails are considered as serious pests in filed crops cella vestalis and in the slug Lehmannia marginata, in many parts of the world. They result in damage and respectively. Moreover, the digestive gland was re- great losses in various vegetation fields (El-Okda, 1981; ported to be composed of two cell types only in both Gabr et al., 2007; Ramzy, 2009). Zinc oxide (ZnO) con- the land snail Eobania vermiculata (Zaldibaretal., siders one of the most used types of metal-based NPs, 2007)andtheslugLimax maximus (Abdel-Haleem & with the third largest annual production in volume EI-Kassas, 2013). Mohammad et al. The Journal of Basic and Applied Zoology (2021) 82:34 Page 6 of 9

Fig. 4 Photomicrograph of sections through the salivary gland of a untreated slugs showing three types of cells: (I) purple colored cells, (II) blue colored cells, and (III) vacuolated cells. b Slugs treated with 0.01 ZnO NPs showing: empty cells(EC). c Slugs treated with 0.02 ZnO NPs showing acidophilic cells with flattened marginal nuclei (arrows). d Slugs treated with 0.05 ZnO NPs showing spaces between cells (arrows)

In slugs and snails, the digestive gland is the major site that the salivary glands of L. maximus possess three for metal accumulation (Berger & Dallinger, 1993; Mari- types of cells. In contrast, Abdel Gawad et al. (2018) re- go’mez et al., 2002), and plays a critical role in metal de- ported four different cell types comprised mucocytes I, toxification (Kammenga et al., 2000). In the present mucocyte II, vacuolated cells, and granular cells in saliv- study cytoplasmic vacuolation and degeneration was no- ary gland of Eobania vermiculata. ticed in the digestive acini of treated slug Lehmannia The present histological observations in the cells of nyctelia. These results are in accordance with that ob- the salivary gland of Lehmannia nyctelia treated with tained by Abdel-Haleem and EI-Kassas (2013) who stud- ZnO NPS revealed cytoplasmic vacuolation and disinte- ied the effect of three plant extracts on maximus. gration of the digestive tubules epithelial cells. These Additional histological changes such as tubular disrup- findings are similar to the results reported by Mustafa tion in the digestive tubules were observed. Sharaf et al. and Awad (2018) who found vacuolated cytoplasm and (2015) reported the same result in the digestive gland of degenerated nuclei in the cells of salivary gland of slug land snail Helicell vestalis treated with some pesticides. Limax maximus after treatment with thymol. Bour et al. (2015) suggested that when NPs are In the present work, the histological alterations when ingested, they may get stuck in the digestive tract and ovotestis of the Lehmannia nyctelia is exposed to ZnO not be absorbed but promote physiological changes that NPs, the disruptions within acini and gametic cells were cause damage to the organism, such as a decrease in the observed. Elsewhere, some alterations were observed absorption of nutrients. Hooper et al. (2011) raised the such as, spermatocytic degeneration, disruptions of oo- possibility that a fraction of Zn accumulated in Eisenia cytes, and appearing fibrosis in the lumen of acini. veneta organisms through NPs is present in the nano- Wangsomnuk et al. (1997) noticed the same results in form, remaining intact inside the cell but still affecting Indoplanorbis exustus. Additionally, Zhou et al. (1993) its metabolism. noticed reduction in the number of spermatozoa and oo- In the present study, the salivary glands of Lehmannia cytes in Biomphalaria glabrata after the exposure to nyctelia revealed that there are three different cell types: niclosamide. type (I) purple colored cells, type (II) blue colored cells, Dimkpa et al. (2011) and Heggelund et al. (2014) de- and type (III) vacuolated cells. These results are in ac- cided that accumulation of NPs causes disorders in the cordance with Mustafa and Awad (2018) who revealed cells which led to toxicity through the formation of Mohammad et al. The Journal of Basic and Applied Zoology (2021) 82:34 Page 7 of 9

Fig. 5 a Photomicrograph of sections through the gonads shows: spermatocyte (SC), spermatids (S), numerous spermatozoa (SP), growing oocytes (GO), and vitellogenic oocytes (VO). b Slugs treated with 0.01 ZnO NPs showing fibrosis (Fi), blood cells infiltration (FL), space (S), and the simple squamous cells layer ruptured in some positions (arrows). c Slugs treated with 0.02 ZnO NPs showing empty spaces (S) and fibrosis (stars) and d slugs treated with 0.05 ZnO NPs showing fibrosis (Fi) and aggregation of early gametogenesis stages(arrows) reactive oxygen species (ROS). Pinto et al. (2003) re- Committee’s reference number vealed that the reactive oxygen species (ROS) has toxic Not available effect in the biological system, which disrupts normal Authors’ contributions cellular processes. WAM, SMA, SMS, and NMF designed the practical part of the work. NMF The present histopathological examinations indicated prepared, characterized ZnO NPs, and analyzed data obtained from that, ZnO NPs induced toxicity in cells of digestive nanoparticles characterization. WAM and SMS collected animals from the field and carried out the animal experiment. SMA carried out the data gland, salivary glands, and hermaphroditic gland in Leh- analysis, prepared figures, and wrote the manuscript. All authors read and mannia nyctelia. approved the final manuscript.

Funding Conclusion Not applicable In conclusion, ZnO NPs caused denaturation of the ani- Availability of data and materials mal organs which lead to abnormal functions of these Data generated during this study are included in this published article. organs and finally affect its fitness. The extensive use of Please contact authors for data. ZnO NPs will lead to destructive effects on animals and will pollute the environment. The present study indi- Declarations cated that Lehmannia nyctelia can be used as bio- Ethics approval and consent to participate indicator to the pollution of the terrestrial environment. All procedures and experiments with animals in this study were approved by the Animal Care and Use Committee at the National Health Research Institute, Egypt. Abbreviations ZnO NPs: Zinc oxide nanoparticles; ROS: Reactive oxygen species; Consent for publication FTIR: Fourier transform infrared spectroscopy; FTIR: Fourier transform infrared Not applicable spectroscopy; SEM: Scanning electron microscopy; TEM: Transmission electron microscopy; UV-visible spectroscopy: Ultraviolet-visible spectroscopy Competing interests The authors declare that they have no competing interests. Acknowledgements The authors would like to express their thanks to Prof. Dr. Ahmed Hamed, Author details Prof. Dr. Naser El Shimy, and Dr. Torkia Abul-Maged for their continuous help 1Zoology Department, Faculty of Science, New Valley University, El-Kharga, and advice. Egypt. 2Zoology Department, Faculty of Science, Assiut University, Assiut, Mohammad et al. The Journal of Basic and Applied Zoology (2021) 82:34 Page 8 of 9

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